Research conducted in the Biochemistry of Proteins Section is focused on basic mechanisms and regulation of protein degradation in bacterial and human cells. Controlled intracellular protein degradation is vitally important, serving both regulatory and protein quality control functions. Most intracellular protein degradation is carried out by multi-component, self-compartmentalized ATP-dependent proteases, which selectively screen potential targets, control access to sequestered proteolytic sites, and can generate discrete degradation products that are recycled to amino acids or sometimes serve as signaling messengers and activators of various cellular responses. Our efforts have been directed toward characterization of the structural and biochemical properties of the ATP-dependent Clp and Lon proteases. In the last year, progress has been made in several areas. We have (in collaboration with Dr. Bijan Ahvazi, NIAMS) refined and published the crystal structure of human mitochondrial ClpP, which has provided the basis for a unique model for the role of the N-terminal peptide of ClpP in regulating or facilitating the passage of substrates through the translocation channel into the active site chamber. Mutagenesis studies have shown that deletion of more than one amino acid from the N-terminus leads to a dramatic decrease or total loss of ClpP activity. In other studies done in collaboration with Dr. Ann Ginsburg, NHLBI, we have shown that human ClpP forms stable heptamers that require the chaperone component, ClpX, to assemble into the bilayered structure with sequestered active sites. These data suggest that assembly of ClpP may be regulated in human cells as a means of controlling the amount or the specificity of ClpP activity. We have obtained stable human cancer cell lines over-expressing either active or inactive mutant forms of human ClpP. Preliminary data suggests that excess human ClpP has effect on the timing or extent of cisplatin-induced apoptosis. We will examine changes in the level of pro- and anti-apoptotic proteins in mitochondria in response to altered expression of ClpP. Efforts are underway to manipulate the cellular content of human ClpP using siRNA techniques and to determine the role of human ClpX on the cellular responses to ClpP so far observed. Biochemical studies of ClpAP and ClpXP have provided several intriguing insights regarding substrate binding by the chaperone component. Short peptides containing sequence motifs recognized by ClpA or ClpX have been shown to bind with a stoichiometry of one peptide per hexamer. Interesting, peptides with different sequence motifs recognized by ClpA compete for binding, indicating that peptide interaction sites may be deformable and adaptable to different motifs or that the sites are structurally complex and contain multiple docking sites that bind different motifs. Because ClpA is a six-fold symmetric complex, limiting binding to one peptide requires a mechanism to exclude peptides from the remaining equivalent sites following binding of the first ligand. We are investigating whether Clp chaperones undergo a conformational change on peptide binding to explain negative cooperativity of binding or whether the peptide binding sites lie close together near the center of the ring and either overlap or sterically interfere with each other. Studies with the adaptor protein, ClpS, which can alter the substrate preference of ClpA, show that ClpS exerts its effect on ClpA also at a stoichiometry of one ClpS per hexamer. These data combined with earlier crystal structure data lead to a hypothesis that the chaperone subunits, though identical, cannot assemble into tightly bonded symmetrical complexes and must undergo some asymmetric conformation change to allow formation of a closed planar ring. Induced asymmetry may be necessary to allow the forces exerted on the unfolded substrates to be of unequal magnitude or to be applied at separate times providing a means of vectorial translocation of the extended polypeptide through the chaperone. Analysis of ClpA bound to substrates is being carried out in two ways. We have created mutants of ClpA into which we can introduce structural probes to obtain information about conformational changes and internal distances in the complexes and distances between bound ligands and sites in the protein. Cross-linking experiments are also contributing to identification of residues in the vicinity of substrate interaction sites. Preliminary cross-linking data indicates that the initial peptide binding site is in the large AAA subdomain of ClpX and ClpA-NBD1. Also, in collaboration with Dr. Di Xia, LCB, NCI, crystals of ClpA with peptide substrate bound or in a complex with the 70 kDa protein, RepA, have been obtained. Optimization of methods to obtain high resolution diffraction quality crystals is underway. We have obtained a crystal structure of ClpP with a peptide covalently linked at the active site. This structure provides the first view of the interaction of substrates in the peptide binding groove within the ClpP chamber. This work is still in progress, and we are extending these studies by the use of mutants that have low catalytic turnover rates in order to co-crystallize longer peptide substrates with ClpP to map the interaction sites on both sides of the scissile bond. Studies with Lon protease have provided the first ever structural data on the N-terminal domain. We have found that the first 120 residues of Lon adopt a unique fold and are currently extending the structural analysis by crystallizing a more complete N-domain containing the predicted coiled-coil region. Lon protease is activated in vivo by interaction with several polymers, including polyphosphate and some nucleic acids. We are currently mapping the domain of Lon responsible for interaction with these polymers and will try to visualize the complexes of Lon with them by cryo electron microscopy in collaboration with Dr. Alasdair Steven, NIAMS.Budget for 2003-2004 634,594